1. Field of the Invention
The present invention relates to determining the prognosis of a patient with breast cancer by determining whether the HER-2/neu gene is amplified in tumor cells.
2. Description of Related Art
Breast cancer remains a major cause of illness and death among women in the United States, with over 180,000 new cases and 44,000 deaths per year (American Cancer Society, 1997). Possibly the most important predictor of clinical course in breast cancer is the presence or absence of lymph node metastases. Many prognostic indicators aid in evaluation of invasive cancers in addition to the presence or absence of lymph node metastasis, including tumor size, histologic type, tumor grade (differentiation reflected in extent of gland formation), nuclear grade (extent of nuclear alteration and frequency of mitosis), DNA content (ploidy), and hormone receptor status. A reasonable and desirable approach would be the use of prognostic factors to risk-stratify invasive breast cancer patients into low-risk and high-risk groups in terms of the probability of recurrence (McGuire, el al., 1990).
The HER-2/neu (ERBB2) gene is an oncogene which shares significant homology to the epidermal growth factor receptor (EGFR) gene (Yamamoto, et al, 1986) and the retroviral gene v-erbB. It was first detected as a mutated transforming gene in chemically induced rat neuronal tumors. It has been isolated from diverse sources, including: rat neuroblastoma (Schechter et al, 1985); human tumor lines from gastric cancer (Fukushige et al, 1986); salivary adenocarcinoma (Semba et al, 1985); and a human breast cancer cell line where HER-2/neu was identified in an amplified form (King et al, 1985). The gene has been localized to 17q11.2q12 (Human Gene Mapping 11, 1991), in a region where several genes relevant to breast cancer are located, including BRCA1 estradiol-17xcex2 dehydrogenase, NM23 and RARA.
Current evidence indicates that HER-2/neu protein over expression and gene amplification are indicative of poor patient prognosis at all stages of breast tumor development. Amplification appears early in tumor progression (Iglehart et al 1990 and Van de Vijver et al 1988), and when present is homogeneously distributed throughout the tumor (Press et al, 1994). Thus, it is a logical choice as a prognostic marker when used as an adjunct with other accepted prognostic indicators.
While such immunoassays for Her-2/neu protein have been commercially available, interpreting results is somewhat difficult. Protein denaturation or degradation during handling, staining embedding in paraffin and sectioning gives variable results, including both false negatives and false positives. Additionally, slightly different conditions during antibody-antigen binding results in false positives and false negatives. Unacceptable results have been reported for immunohistochemical detection of HER-2/neu amplification. See Thor et al 1989; Richner et al, 1990; O""Reilly et al, 1991; and Loveldn et al, 1991. By contrast counting the number of copies of the HER-2/neu gene in a cell represents a more objective determination and involves DNA markers which are less susceptible to degradation and provide less variable results.
HER-2/neu gene amplification status is useful as an adjunct in the evaluation of the prognosis of node negative breast cancer patients and is also an independent marker of high risk in node negative patients. Amplification of HER-2/neu is indicative of poor patient prognosis at all stages of breast cancer development and correlates with relatively shorter disease-free and overall survival.
Studies have shown positive correlation between HER-2/neu gene amplification and other common indicators of poor prognosis in breast cancer (Tsuda, et al, 1989 and Seshadri, et al., 1993 and Slamon et al, U.S. Pat. No. 4,968,603). However, even strong breast cancer prognostic factors, such as number of positive lymph nodes, tumor size and histograde do not predict patient outcome unfalteringly (Wright, et al., 1989 and Ro, et al., 1989). Current evidence indicates that HER-2/neu protein over expression and gene amplification are indicative of poor patient prognosis at all stages of breast cancer development (Seshadri, et al., 1993, Wright, et al., 1989 and Niehans et al., 1993). Because HER-2/neu amplification appears early in breast cancer progression (Iglehart, et al., 1990 and van de Vijver, et al., 1988) and, when present is homogeneously distributed throughout the cancer (Iglehart, et al., 1990 and Press, et al., 1994), it can serve as a prognostic marker for this disease (when used as an adjunct with other accepted prognostic indicators).
The use of Fluorescent In-Situ Hybridization (FISH) targeted to the HER-2/neu gene, has successfully demonstrated gene amplification in breast cancer cell lines and primary tumors, and has shown that FISH results are concordant with other measures of amplification (Kallioniemi, et al, 1992). [The gene has been localized to 17q11.2-q12 (Human Gene Mapping 11, 1991), in a region where several genes relevant to breast cancer are located, including BRCA1, estradiol-17 dehydrogenase, NM23, and RARA.] FISH technology combines the advantages of direct gene amplification assessment with direct localization in morphologically identified tumor cells. FISH is applicable to tumors of all sizes because studies can be performed on sections from the original specimen blocks used for diagnosis. In many samples, direct comparison can be made with FISH assays on normal cells from the same preparation. Further, if amplification were localized rather than diffusely distributed within a tumor, it would be detectable by FISH but could be diluted below detectable limits in extracted tumor DNA required for other procedures.
When performing an assay of such importance to the patient, it is critical to have appropriate controls. Sections of previously tested tissue are somewhat undesirable as controls due to cell variability, unclear boarders, necrotic tissue in the center of the tumor, variable responses to protease digestions and finite source material. Therefore, there is a need for quality control materials which can be run with every test which lack the above mentioned problems.
There is also a need for a set of statistical benchmarks to allow the medical practitioner to stratify the patient according to likelihood of cancer recurrence. This will aid the practitioner and patient in deciding whether aggressive treatments (e.g. chemotherapy, radiation and anti-HER-2/neu therapy) should be employed in lieu of a passive xe2x80x9cwatchful waitingxe2x80x9d approach.
The present invention is directed to methods and reagents which determine the number of copies of the HER-2/neu gene in a breast cancer specimen. This method uses a FISH assay for HER-2/neu in surgically removed breast cancer tissue. Determination of an abnormally high copy number of the gene correlates with poor prognosis and such patients should be treated aggressively.
The present invention is also directed to a set of control slides, one of which has a normal copy number of the HER-2/neu gene, one has a high copy number of the HER-2/neu gene and one has a slightly elevated copy number of the HER-2/neu gene.
The present invention further includes the preparation of control slides using cell lines instead of primary tumor tissue. The preferred cell lines used for controls are one with high amplification of the HER-2/neu gene, one with non-amplification and one with low amplification.
The HER-2/neu gene detection system of the present invention is a kit consisting of DNA probe and detection reagents that yields a green fluorescent signal at the site of each HER-2/neu gene, on a blue fluorescent background of stained nuclear DNA The kit is intended to be used with sections (4 xcexcm) of formal fixed, paraffin-embedded human breast cancer tissue. The kit is untended to include or recommend the use of another kit which includes the control lines.
The HER-2/neu gene detection system of the present invention is preferably a fluorescence in situ hybridization (FISH) DNA probe assay that determines the qualitative presence of HER-2/neu gene amplification on formalin-fixed, paraffin-embedded human breast tissue as an aid to stratify breast cancer patients according to risk for recurrence or disease-related death. It is indicated for use as an adjunct to existing clinical and pathologic information currently used as prognostic indicators in the risk stratification of breast cancer in patients who have had a primary, invasive, localized breast carcinoma and who are lymph node-negative.
A recent review and comparison is Ross et al, The Oncologist 3: 237-252 (1998).